Lentiviruses are common vectors used in gene therapy because they can transduce non-dividing cells and offer stable integration into a target cell's genome. The host range of lentivirus vectors can be altered by pseudotyping with glycoproteins derived from enveloped viruses. Current gene therapy typically employs lentiviral vectors pseudotyped with the VSV-G envelope protein (VSV-Gpp), which has a ubiquitous host cell receptor, thereby allowing transduction of most cell types. However, VSV-G itself is known to be cytotoxic and the envelope cytotoxicity limits the amount of VSV-Gpp that can be concentrated and used for cell transduction. That is, while VSV-G envelope has great stability in the vector particle, and can be concentrated to high titers via ultracentrifugation, the toxicity of VSV-G itself limits the viral titer that can be used as too high a concentration of VSV-Gpp applied to the target cell population results in apoptotic cell death. In addition, because it has a ubiquitous host cell receptor, VSV-Gpp cannot be targeted to specific populations of cells. Additionally, when VSV-Gpp is administered intravenously to mice, the majority is trapped in the liver, sometimes termed the “liver sink” effect, which is detrimental to the gene therapy unless the desired target cells reside in the liver.
To overcome these shortcomings of VSV-Gpp, other strategies have been devised for targeted lentiviral gene therapy. One common strategy involves pseudotyping lentiviral vectors with a modified Sindbis virus envelope that has been mutated to remove its own receptor binding site and engineered to display a “ZZ” motif from proteinA—a motif that binds to the Fc region of most antibodies. Incubation of the Sindbis-ZZ pseudotyped vectors with a specific monoclonal antibody theoretically should target the lentiviral particles to the cell-type in question. (See Morizono K et al., 2005, Nat Med Vol 11(3):346-52). However, while the technique works well in vitro, in vivo the majority of the intravenously administered Sindbis-ZZ pseudotyped vector is still trapped in the liver, regardless of the antibody used. As such, improved methods of overcoming the shortcomings of VSV-Gpp are still needed.
Nipah virus (NiV) is an emerging paramyxovirus that causes acute fatal encephalitis. Two envelope glycoproteins (the fusion and attachment glycoproteins) mediate cellular entry of Nipah virus. The attachment protein, NiV-G, functions in recognition of the receptor (EphrinB2 and EphrinB3). Binding of the receptor to NiV-G triggers a series of conformational changes that eventually lead to the triggering of NiV-F, which exposes the fusion peptide of NiV-F, allowing another series of conformational changes that lead to virus-cell membrane fusion. EphrinB2 was previously identified as the primary NiV receptor (Negrete et al., 2005), as well as ephrinB3 as an alternate receptor (Negrete et al., 2006). In fact, NiV-G has an extremely high affinity for ephrinB2 and B3, with affinity binding constants (Kd) in the picomolar range (Negrete et al., 2006) (Kd=0.06 nM and 0.58 nM for cell surface expressed ephrinB2 and B3, respectively). Significantly, residues important for ephrinB2/B3 interactions with their endogenous ephB receptors are also critical for their activity as NiV receptors, indicating that the NiV attachment glycoprotein (NiV-G) can block endogenous ephrinB2-ephB4 receptor interactions.
Ephrin receptor-ligand pairs (Eph-ephrin) are membrane associated receptor tyrosine kinases (RTKs) with well-established roles in development; they regulate cell boundaries during tissue formation, and provide guidance cues during neurogenesis and angiogenesis. (See Pasquale E B. Eph-ephrin bidirectional signaling in physiology and disease. Cell. 2008; 133:38-52.) Cognate interactions activate both the Eph receptor (forward signaling) and ephrin ligand (reverse signaling) on opposing cells. These bi-directional signaling cascades result in cell-cell repulsion or attraction, depending on cell type or other microenvironmental cues.
EphrinB-ephB receptor-ligand interactions are a common regulator of multiple somatic stem cells, e.g., intestinal crypt stem cells and hematopoietic stem cells (Pasquale (2008) Cell 133:38-52; Poliakov et al. (2004) Dev. Cell. 7:465-480), where differentiation is a carefully choreographed molecular and cellular response to local environmental determinants. EphrinB2, in particular, has been identified as a molecular stem cell signature common to human embryonic, neural, and hematopoietic stem cells (hESC, hNSC and hHSC) (Ivanova et al. (2002) Science 298:601-604). Its cognate receptor, EphB4, has also been shown to affect mouse ESC fate. Despite much evidence from model systems that ephrinB2/ephB4 axis may be intimately involved in ESC fate (survival, self-renewal, and pluripotency), this particular axis has not been carefully studied in human ESC.
In mouse ESC, ephB4 inactivation results in bias against differentiation: ephB4-deficient mouse ESCs appear to remain in a more primitive state and are impaired in embryoid body (EB) formation in general and mesodermal differentiation in particular. (Wang et al. (2004) Blood 103:100-109)). Conversely, over expression of ephB4 in umbilical cord blood CD34+ cells results in a loss of the most primitive progenitors (LTC-ICs and CD34+/CD38− cells) likely due to differentiation into more committed precursors. (Wang et al. (2002) Blood 99:2740-2747)). EphrinB-ephB ligand-receptor interactions are promiscuous, and the lack of highly specific yet versatile reagents to interrogate this axis has hampered the understanding of ephrinB2/ephB4's role in hESC fate (pluripotency, survival and self-renewal) and HSC lineage commitment. Understanding the regulation of this signaling axis could improve the culture of hESCs and the efficiency of HSC lineage differentiation, both previously key barriers in the field.
EphB4 and ephrinB2 are both expressed in ESC and likely contribute to some aspect of stem cell fate. However, while ephrinB2 is clearly also involved in ectoderm and endoderm differentiation, ephB4 is unique amongst ephB receptors for not being expressed in the central nervous system. Thus, ephrinB2 “reverse” signaling and ephB4 “forward” signaling likely play overlapping but distinct roles in germ layer commitment and differentiation. Understanding the relative contribution of each signaling pathway may result in more optimal conditions for directing the differentiation of specific cell types.
Finally, ephrinB-ephB usually follows a gradient of ligand-receptor interactions, and expression of ephrinB2 is indeed heterogeneous within an ESC colony. Understanding the basis for the heterogeneity seen in human ES cell cultures will lead to more robust culture conditions that give rise to more homogenous population of cells suitable for regenerative medicine.
Eph-ephrin RTK expression is dysregulated in multiple cancers, and various members of this RTK family have been implicated in cancer development, progression, and subsequent metastases (See Pasquale E B. Eph receptors and ephrins in cancer: bidirectional signaling and beyond. Nat Rev Cancer. 2010; 10:165-180).
Deciphering the role of Eph signaling activities in cancer is confounded by the promiscuity of interactions between Eph-ephrin receptor-ligand pairs, and the complexity of the resultant signaling cascades. Nevertheless, the centrality of ephrinB2 in facilitating tumor angiogenesis and promoting invasion and metastasis is supported by a slew of studies that provide a sound mechanistic basis for its action (See Pasquale E B. Eph receptors and ephrins in cancer: bidirectional signaling and beyond. Nat Rev Cancer. 2010; 10:165-180). As such, soluble EphB4 inhibits tumor growth in multiple xenograft models (see Kertesz N, Krasnoperov V, Reddy R, et al. The soluble extracellular domain of EphB4(sEphB4) antagonizes EphB4-EphrinB2 interaction, modulates angiogenesis, and inhibits tumor growth. Blood. 2006; 107:2330-2338; Kumar S R, Scehnet J S, Ley E J, et al. Preferential induction of EphB4 over EphB2 and its implication in colorectal cancer progression. Cancer Res. 2009; 69:3736-3745; Spannuth W A, Mangala L S, Stone R L, et al. Converging evidence for efficacy from parallel EphB4-targeted approaches in ovarian carcinoma. Mol Cancer Ther. 2010; 9:2377-2388), while molecular genetic evidence implicates ephrinB2 reverse signaling in the activation of VEGFR2 that leads to vessel sprouting (See Branco-Price C, Johnson R S. Tumor vessels are Eph-ing complicated. Cancer Cell. 2010; 17:533-534; Sawamiphak S, Seidel S, Essmann C L, et al. Ephrin-B2 regulates VEGFR2 function in developmental and tumor angiogenesis. Nature. 2010; 465:487-491). The latter point suggests the exciting possibility that blocking ephrinB2 signaling may synergize with anti-VEGF therapies. Furthermore, amongst all the ephrins examined, only ephrinB2 on stromal cells (fibroblast, endothelial cells, or pericytes) activates ephB3/ephB4 on invasive prostate cancer cells leading to loss of contact inhibition of locomotion (CIL), the tumor invasive phenotype responsible for cancer metastases (See Astin J W, Batson J, Kadir S, et al. Competition amongst Eph receptors regulates contact inhibition of locomotion and invasiveness in prostate cancer cells. Nat Cell Biol. 2010; 12:1194-1204; Wang B. Cancer cells exploit the eph-ephrin system to promote invasion and metastasis: tales of unwitting partners. Sci Signal. 2011; 4:pe28).
Use of Nipah virus in conjunction with a lentivirus vector has heretofore been hampered by the fact that paramyxoviral envelopes are known not to pseudotype functionally onto lentiviral particles, presumably due to some incompatibility of the cytoplasmic tail of the fusion and attachment glycoproteins with the matrix (gag) protein of HIV.
There remains a need for improved gene therapy compositions and methods that allow for enhanced delivery of the gene product to the target cells or tissues.